JP2627896B2 - Myristyl compounds, their preparation and use - Google Patents

Description

The present invention relates to an amino acid and a peptide having a myristylglycine residue, a method for producing the same and a use thereof, and in particular, to an agent for transforming a virus-infected cell or using the above amino acid or a peptide as an active ingredient. Things.

BACKGROUND OF THE INVENTION AND PRIOR ART In general, it is known that the amino terminus of a protein is blocked by acetyl, pyroglutamyl and formyl groups.
Et al. Have found myristic acid (long chain fatty acid) covalently linked to its amino terminus in the catalytic subunit of cAMP-dependent protein kinase (Proc. Nat).
I. Acad. Sci. USA, Vol. 79, 6123-6131, 1982). This myristyl-terminal protein has the amino acid sequence Since then, the presence of a terminal myristyl group has been reported one after another in the following various proteins.

Calcineurin B: (A. Aitkan et al, FEBS LETTERS, Vol. 150, No. 2, 314-31
8,1982) NADH- cytochrome b ₅ reductase: (J. Dzols et al, The Journal of Biological Chemistr
y, Vol. 259, No. 21, 13349-13354, 1984) It has recently been reported that some of the constituent proteins of some viruses have the amino terminal myristylated similarly. The most recent report reported that the amino terminus of VP2, the major component of the SV40 virus (Simian Virus40), a well-known polyomavirus derived from rhesus monkey kidney cells, is myristylated. (Streuli et al. Nature V
ol. 326 619-622, 1987). In particular, the present inventors have reported several reports on myristylation of a retrovirus constituent protein that has recently attracted attention among viruses. Retroviruses are a genus of virus that causes adult T-cell leukemia (ATL) and acquired immune deficiency syndrome (AIDS), which have received much attention today. Actively studied in various countries. The present inventors have also studied myristylation of Rous sarcoma virus (RSV) belonging to this retrovirus. The report below relates to myristylation of the protein kinase p60src, a gene product of RSV.

Protein kinase p60src: (AM Schultz et al, Science, Vol. 227, 427-429, 198
5) Recently, myristylation of a constituent protein of HTLV-III / LAV, which is a virus causing acquired immunodeficiency syndrome (AIDS), has also been reported. This report indicates that myristylation has occurred at the amino terminus of the gag protein, a major component of the AIDS virus.
(Sarngadharan et al. Ann.Inst.Pastuer / Virol.138,13
3-166 1987; International Con-ference on AIDS, Par
is, June 23-25, 1986) As shown in the above amino acid sequences, in these reports, a myristyl group, that is, CH _3- (CH ₂ ) ₁₂ -CO
The group has been shown to be attached to the amino terminal glycine residue. However, these reports
All of them merely describe myristylation from the viewpoint of structural analysis of virus constituent proteins, and do not mention at all the relationship between such a structure and the transformation of virus-infected cells or virus growth.

The present inventors have conducted various studies on myristylation in retroviruses using Rous sarcoma virus (RSV), a type of retrovirus. In the process, myristylglycine and its oligopeptide derivatives were synthesized, their virus growth inhibitory effects and the like were examined, and it was confirmed that these substances had the effect of suppressing the transformation of cells infected with the virus. Japanese Patent Application No. 61-257823 (JP-A-63-146851). That is, in this earlier application, the present inventors have developed a novel and effective method for myristylglycine and its peptide derivatives (ie, myristylglycyl oligopeptide), which may be useful for biological analysis of the amino-terminal myristyl group of the RSV gene product. In addition to developing synthetic methods, they have further found that these synthesized myristylglycine or oligopeptide derivatives thereof have an inhibitory effect on the transformation of virus-infected cells.

Object of the Invention Based on the above facts, the present inventors have further studied myristylation in a retroviral gene product in order to obtain a substance capable of suppressing the transformation or virus growth of virus-infected cells. Was. As a result, as described in the specification of Japanese Patent Application No. 61-257823 (Japanese Patent Application Laid-Open No. 63-1468).
51), the myristylglycine and the oligopeptide having a myristylglycine residue are reconfirmed to be effective in suppressing the growth of a retrovirus, and other amino acids and peptides having a myristylglycine residue are also retroviruses. Have been found to exhibit a growth inhibitory effect, and have completed the present invention.

Structure and Effect of the Invention The myristyl compound according to the present invention is characterized by having a myristylglycine residue at the amino terminal and an aldehyde group at the carboxy terminal.

The mechanism by which the myristyl compound (amino acid and peptide) of the present invention suppresses the transformation of cells infected with a virus, particularly a retrovirus, and the growth of the virus has not been elucidated. Myristylation of terminal glycine residues plays an important role in virus formation during virus growth or in the synthesis of viral gene products in virus-infected cells,
The myristyl compound according to the present invention is considered to inhibit myristylation of a virus-constituting protein by a myristyl group characteristic of the compound, and further specifically myristylates a viral gene product by an amino acid or peptide containing a myristylglycine residue. It is considered that it can be suppressed.

Among myristylglycinals or oligopeptide derivatives thereof which are preferred embodiments of the present invention, those having a relatively small molecular weight are preferred as the preferred structures having a high virus growth inhibitory effect. One example is myristylglycinal, which has the most basic structure of the present invention, and its oligopeptide derivative is a relatively short oligopeptide derivative containing about 2 to 4 amino acids in the peptide. Can be The amino acid sequence that binds to the myristyl group in the oligopeptide derivative of the present invention depends on the target virus when used as a virus-infected cell transformation or virus growth inhibitor,
It is possible to select a preferred sequence. That is,
The amino acid sequence of the oligopeptide derivative of the present invention corresponding to the target virus can be inferred from the amino acid sequence of a protein which is considered to undergo myristylation among viral gene products, and the like, by a specific amino acid sequence.
For example, the p60 in RSV we studied
Since the amino acid sequence of the protein subjected to myristylation of src is (Myr-) Gly-Ser-Ser-Lys-Ser-, this amino acid sequence is used for the transformation of virus-infected cells for RSV or the virus growth inhibitor. The amino acid corresponding to the above is bound to myristylglycine if necessary to obtain the myristylglycyl peptide of the present invention or a derivative thereof.

From the previous studies by the present inventors, the following sequence is an example of a preferred form of the substance of the present invention when targeting a retrovirus.

1) Myr-Glycinal 2) Myr-Gly-Glycinal 3) Myr-Gly-Gly-Glycinal 4) Myr-Gly-Ser-Glycinal 5) Myr-Gly-Ala-Glycinal (Myr- is a myristyl group peptide-bonded to an amino acid) The myristyl compounds of 1), 2) and 3) are effective not only for a specific virus but also widely and generally, and the peptide of 4) is particularly effective for RSV which has been tested by the present inventors. . The myristyl compound of 5) has a sequence corresponding to the gag protein of HTLV III / LAV among human retroviruses.

The present invention further provides a method for producing a myristyl compound that is used as a transformant for virus-infected cells or a virus growth inhibitor.

Hereinafter, a method for producing myristylglycinal and its oligopeptide derivative will be described in detail.

The myristic acid chloride used in the synthesis of myristylglycinal and its oligopeptide derivative of the present invention can be prepared from commercially available myristic acid by an ordinary method. Also, when preparing an acid chloride of myristyl amino acid, particularly myristyl glycyl chloride, or myristyl peptidyl chloride, the desired amino acid can be easily prepared by binding the required amino acid to this myristic acid by a conventional method, for example, an acid-chloride method. Myristyl amino acids or myristyl peptides can be prepared. Of course, if the desired myristyl amino acid or myristyl peptide is available as a commercial product, these can also be used.

Preferred examples of the myristyl peptide used herein include those in which one or two amino acids are peptide-bonded to the myristyl group.

On the other hand, commercially available aminoacetaldehyde diethyl acetal can be prepared at an appropriate concentration and used.

The alkali used as a catalyst in the present invention, any suitable alkali, for example _{NaHCO 3, Na 2 CO 3,} NaOH, there is Na ₃ PO ₄ or the like, which are about 0.1 to 1.0 molar, preferably in a reaction solvent Use at 0.3-0.6 molar concentration.

In the present invention, polar solvents which are important factors for obtaining the target myristylglycinal or oligopeptide derivatives thereof include nitrile solvents such as acetonitrile and propionitrile; ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone; Can be used. However, alcohols are the same polar solvents, but are not used in the present invention because they are active against myristin chloride. That is, the polar solvent used in the present invention must be inert to each starting reactant. Preferred polar solvents are nitrile solvents, especially acetonitrile. The polar solvent used can be used as a reaction solvent in the form of 100%, but is preferably in the form of an aqueous solvent mixed with water, and the polar solvent is about 10 to 70% by weight, preferably 20 to 50% by weight of water. % Used in the presence form.

These reactions can be carried out by a usual method such as an acid-chloride method. That is, the reaction can generally be carried out at room temperature or lower under stirring conditions, and the reaction time is about 6 to 20 hours. The product obtained after completion of the reaction can be obtained as a high-purity product by subjecting it to ordinary purification treatment such as extraction with ether or the like, recrystallization with alcohol or the like. As an example of a preferred reaction, a solution of aminoacetaldehyde diethyl acetal dissolved in a suitable buffer solution is myristic acid chloride,
Myristyl glycyl chloride or myristyl peptidyl chloride was added and reacted in an acetonitrile solution.After the reaction was completed, the reaction mixture was extracted with ether or the like and then recrystallized with hot ethanol or the like, and then recrystallized with hot ethanol or the like. Obtain cinal diethyl acetal.

The thus-obtained myristylglycinal diethylacetal or the diethylacetalized aldehyde-modified myristyl peptide is treated with trifluoroacetic acid to obtain the myristylglycinal or aldehyde-modified myristyl peptide of the present invention. However, since these aldehyde-ized myristyl compounds are unstable, it is necessary to protect the aldehyde group usually with an acetal group and remove the acetal group by the above reaction immediately before use.

The thus-obtained myristylglycinal or oligopeptide derivative thereof is excellent in the effect of suppressing the growth of viruses, particularly the effect of suppressing the growth of retroviruses. The present inventors have reported that fibroblasts infected with Rous sarcoma virus (RSV temperature mutant tsNY68), a type of retrovirus whose temperature changes significantly with temperature, are normal cells at 41 ° C. and 37 ° C. To transform into cancer cells,
It has the function of returning to normal cells again at 41 ° C. ) Was used to examine the effect of the myristylglycinal of the present invention and its oligopeptide derivative on the transformation of the cells. At the same time, as a comparative experiment, a lauryl group (12 carbon atoms) other than a myristyl group (14 carbon atoms), a palmityl group (16 carbon atoms) and a stearyl group (18 carbon atoms)
The virus growth inhibitory effect of glycinal derivatives having the above was also investigated. As a result, the glycinal derivative having a lauryl group other than myristyl group, palmityl group and stearyl group had almost no effect of suppressing the transformation of virus-infected cells, whereas the glycinal derivative of the present invention having myristyl group was a retroviral derivative. It was found that the proliferation process was significantly suppressed. In this case, the amount of myristylglycinal and its peptide derivative added as an agent for transforming retrovirus-infected cells or as a virus growth inhibitor is about 5 to 50 μmol per infected cell (10 ⁷ cells).

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1: myristyl glycidyl Sinar preparation of myristic acid _{(CH 3 (CH 2) 12} -COOH) of 1g were placed in a test tube, acid added oxalyl chloride 3 ml - performing chloridation. The reaction was left overnight at room temperature to perform a sufficient reaction. After completion of the reaction, excess oxalyl chloride was removed under reduced pressure to obtain myristic acid chloride. In 20 ml of 0.5 M NaHCO ₃ solution, _{3 to 5} ml of aminoacetaldehyde diethyl acetal, 23
To a solution containing 35 mmol was added dropwise 5 ml (2.3 to 3.5 mmol) of an acetonitrile solution of myristic acid chloride with vigorous stirring, and the mixture was left overnight at room temperature (about 20 ° C.) with continuous stirring. The reaction solution was extracted three times with ether (20 ml), and the ether layer was washed three to five times with 10 ml of 1N HCl. After the ether layer was evaporated to dryness, the obtained powdery solid was recrystallized three times with hot ethanol to obtain 600 mg of myristylglycinal diethyl acetal as needle crystals. The melting point of the crystals was about 65 to 67 ° C. as measured by a micromelting point meter (manufactured by Yanagimoto Seisakusho). 1m of this myristyl glycinal diethyl acetal
g was dissolved in 100 µ of trifluoroacetic acid (TFA) and left at 37 ° C overnight to remove the diethyl acetal group, which is a protecting group for the aldehyde group, and to remove excess TFA and ethanol produced by the reaction under reduced pressure. Yielded Myr-Glycinal.

Example 2: Myristyl glycine (Myr-Gly), myristyl diglycine (Myr-G) in place of myristic acid in Example 1.
ly-Gly), myristylglycylserine (Myr-Gly-Se)
Using r), the following three kinds of crystal products were obtained in the same manner as in Example 1. In addition, the melting point of the final product of diethyl acetal was measured in the same manner as in Example 1 and shown below. The oligopeptides used were all
It was obtained as a commercial product from Peptide Research Institute (Osaka). The product melting point Myr-Gly-Glycinal 110~116 ℃ Myr -Gly-Gly-Glycinal 103~105 ℃ Myr-Gly-Ser-glycinal 106~108 ℃ ( melting point, a melting point of diethyl acetal product of the previous compound Example 3: As a comparative reference experiment, an oligopeptide having another fatty acid having a different carbon chain length was prepared in place of the amino-terminal myristyl group. That is, instead of the myristylglycylserine of Example 2, a) laurylglycylserine,
Using b) palmitylglycylserine and c) stearylglycylserine, the following three products were obtained in the same manner as in Example 2. All oligopeptides used were obtained from Peptide Research Institute as commercial products.

a) Lauryl glycylseryl glycinal b) Palmityl glycylseryl glycinal C) Stearyl glycyl seryl glycinal Example 4: Virus growth inhibition test Ten-day-old embryonated chicken egg fibroblasts were prepared by a conventional method. The prepared cells were first cultured at 41 ° C for 48 hours, and then at 37 ° C for 96 hours.
Cultured for hours. When the culture temperature is lowered to 37 ° C (0 hour)
The morphological changes of the cells at the respective culture stages at the time of culturing at 96 ° C. and at 37 ° C. for 96 hours (96 hours) were observed using an inverted microscope, as shown in FIGS. 1a and 1b (control 1 ). That is, in normal cells, cell division and proliferation are observed in both culture stages, and the orientation (orientation) and anchorage dependence (anchorage dependency) are sufficiently observed. Also, RS
V (AsNY60 strain; obtained from the University of Tokyo Institute of Medical Science)
Cells infected at an amount of 1 ml per (× 10 ⁶ ) were cultured in two stages in exactly the same manner as described above, and the results of FIGS. 2a and 2b were obtained by observation with an inverted microscope (control 2). As is evident from these results, in the culture stage at 37 ° C. and 0 hour, as shown in FIG. 2a, the same direction as in FIG. 1a in normal cells was observed, but after culturing at 37 ° C. for 96 hours. (Fig. 2b) shows that the cell division is severe, the direction is lost, and the transformation is clearly occurring.

Further, cells infected with the AsNY68 strain of Control 2 were infected with myristylglycinal prepared in Example 1 (5 × 10 5 cells).
Each of them was added at an amount of 50 μM per ⁶ pieces), and when the temperature was lowered from 41 ° C. to 37 ° C. (0 hour) and the cells were cultured at 37 ° C. for 96 hours, and the morphological changes were observed. And the result of FIG. 3b was obtained. After culturing for 96 hours (Fig. 3b), at the start of culturing at 37 ° C (Fig. 3a)
A substantially similar plasma state was observed, indicating that the orientation and anchorage dependence were sufficiently observed, and that cell division was suppressed. In addition, myristyl glycyl glycal, myristyl glycyl glycyl glycal, and myristyl glycyl ceryl glycinal prepared in Example 2 were added in an amount of 50 μM per infected cell (5 × 10 ⁶ cells), and cultured in the same manner. Similarly, morphological changes were observed. As a result, FIGS. 4a to 4b (myristyl glycyl glycine), FIGS. 5a to 5b (myristyl glycyl glycyl glycal) and FIGS. 6a to 6b (myristyl glycyl glyceryl glycal), respectively. Was obtained. As with myristylglycinal, it can be confirmed that all oligopeptide derivatives suppress cell transformation.

Example 5: Comparative experiment Using lauryl glycyl seryl glycal, palmityl glycyl seryl glycal and stearyl glycyl ceryl glycal as prepared in Example 3, the case of myristyl glycyl ceryl glycal of Example 4 Similarly, the effect on transformation of virus-infected cells was examined. As a result, the results shown in FIGS. 7a to 7b, FIGS. 8a to 8b, and FIGS. 9a to 9b were obtained. However, unlike the myristyl glycinal and oligopeptide derivatives thereof of the present invention, no effect was observed in any case on the transformation of virus-infected cells.

[Brief description of the drawings]

FIGS. 1a and 1b show normal developing chicken egg fibroblasts.
4 is an inverted photomicrograph showing the morphological changes of cells before and after culturing at 37 ° C. for 96 hours (magnification: 150 ×). FIGS. 2a and 2b show RSV-infected embryonated chicken germ cells at 41 ° C.
It is the inverted microscope photograph which shows the morphological change of the cell after culture | cultivation at 37 degreeC and 96 hours at 37 degreeC (0 hour) after culture | cultivation at 37 degreeC (magnification: 150 times). FIGS. 3a and 3b show the morphological changes of the cells at the start of cultivation of RSV-infected embryonated ovary cells supplemented with myristylglycinal at 37 ° C. (0 hour) and after 96 hours of culture. Fig. 4a and Fig. 4b are micrographs (magnification: 150x). Fig. 4a and Fig. 4b show the culture start time (0 hour) of RSV-infected embryonated ovary cells to which myristylglycylglycinal was added (0 hour) and after 96 hours. 5 is an inverted photomicrograph showing the morphological changes of the cells at the same time (magnification: 1
FIGS. 5a and 5b show the morphology of the RSV-infected embryonated hen germ cells supplemented with myristylglycylglycylglycinal at the start of culture at 37 ° C. (0 hour) and after 96 hours of culture. 6a and 6b show an inverted micrograph showing a biological change (magnification: 150 ×), at the time of starting culture of RSV-infected embryonated hen germ cells supplemented with myristylglycylserylglycinal at 37 ° C. H) and an inverted micrograph showing the morphological changes of the cells after 96 hours of culture (magnification: 150 ×). FIGS. 7a and 7b are inverted microscopes showing the morphological changes of RSV-infected embryonated hen oocytes to which laurylglycylserylglycinal was added at 0 and 96 hours after culturing at 37 ° C. It is a photograph (magnification: 150 times). FIGS. 8a and 8b show inverted morphological changes of RSV-infected embryonated hen ovary cells supplemented with palmitylglycylserylglycinal at 37 ° C. after 0 hour and 96 hours. It is a microscope photograph (magnification: 150 times). FIGS. 9a and 9b are inverted microscopes showing the morphological changes of RSV-infected embryonated hen oocytes supplemented with stearylglycylserylglycinal at 37 ° C. after 0 hours and 96 hours. It is a photograph (magnification: 150 times).

Continued on the front page (51) Int.Cl. ⁶ Identification number Reference number in the agency FI Technical display location C07K 1/02 C07K 5/062 5/062 A61K 37/02

Claims (4)

(57) [Claims] 1. A myristyl compound characterized by being selected from myristyl glycinal, myristyl glycyl glycal, myristyl diglycyl glycinal, myristyl glycyl seryl glycal and myristyl glycyl araanyl glycinal. (2) myristic acid chloride, myristyl glycyl chloride, myristyl diglycyl chloride, myristyl glycyl seryl chloride or myristyl glycyl alanyl chloride and aminoacetaldehyde diethyl acetal in an inert polar solvent or in the presence of an alkali; Reaction in an aqueous solvent containing an active polar solvent, characterized in that the resulting compound is subjected to an acid treatment of a diethyl acetalized product, myristyl glycinal, myristyl glycyl glycal, myristyl diglycyl glycal,
Process for producing myristylglycylserylglycinal and myristylglycylalanylglycinal. 3. A myristyl compound selected from myristyl glycinal, myristyl glycyl glycinal, myristyl diglycyl glycinal, myristyl glycyl seryl glycinal, and myristyl glycyl alanyl glycinal as an active ingredient.
An agent for transforming virus-infected cells or inhibiting virus growth. 4. The method according to claim 3, wherein the virus is a retrovirus.